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Kinetics of induction and protective effect of heat-shock proteins after cardioplegic arrest
Kinetics of Induction and Protective Effect of Heat-Shock Proteins After Cardioplegic Arrest Mohamed
Amrani,
M i c h a e l J. D u n n ,
MD, PhD, Joseph
PhD, and Magdi
Corbett, PhD, Samuel
Y. B o a t e n g , BSc,
H. Yacoub, FRCS
National Heart & Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, United Kingdom
Background. Heat-shock proteins are known to enhance cardiac resistance to ischemia. Methods. To evaluate the kinetics of heat-shock protein 70 in relation to its effect on postischemic recovery of cardiac mechanical (cardiac output) and endothelial function (as percentage increase of coronary flow in response to 5-hydroxytryptamine), isolated rat hearts were subjected to prolonged hypothermic cardioplegic arrest at different intervals ranging from 12 to 96 hours after heat stress (n = 6 in each interval). Results. Immunoblotting showed the maximal level of heat-shock protein 70, 0.65 - 0.10 (arbitrary units + standard error of the mean), at 24 hours after heat shock
and similar values at 26 and 30 hours (p = not significant). Postischemic recovery of cardiac output and endothelial function (percentage of preischemic value -+ standard error of the mean) observed at 24 hours was 74.0 +-2.4 and 58.3 -+ 7.2, respectively. Similar values were observed at 26 and 30 hours (p = not significant). Conclusions. In a protocol mimicking conditions for cardiac transplantation, postischemic recovery of cardiac output and endothelial function was improved when the interval between heat stress and ischemia ranged from 24 to 30 hours. This correlated with an apparently critical amount of heat-shock protein 70.
eat-shock proteins (HSPs) belong to a large group of proteins with a wide range of molecular weights. Some HSPs are constitutively expressed, whereas others are induced by a variety of mechanisms but are preferentially expressed after heat shock [1-4]. Heat-shock proteins are known to protect the cell against a further stress. Of particular interest is the finding that HSPs enhance cardiac resistance to ischemia. This has been demonstrated in numerous experimental models of low-flow or regional ischemia using different stress stimuli [4-7]. We ]8, 9] have previously shown a protective effect of heat stress on both endothelial function and mechanical function after cardioplegic arrest. However, the exact time course of induction and the relation between the amount of HSP induced and the protective effect has not been adequately investigated, particularly after cardioplegic arrest [6, 7, 10]. The aim of the present study was to determine the levels of HSP 70 induction at various intervals after heat stress and to identify the amount of HSP affording the maximal recovery of endothelial and mechanical function in a protocol mimicking conditions for cardiac transplantation.
avoid a potential lack of homogeneity related to hormonal cycle. Six hearts were studied in each group. In all studies, animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then placed on a temperature-controlled heating pad (IMS K-Temp control unit; Concleton, Cheshire, England) set at 45°C until body temperature reached 42°C. Body temperature was monitored with a rectal temperature probe and maintained between 42° and 42.5°C for 15 minutes as previously described [10].
H
Material and M e t h o d s Sprague-Dawley rats weighing between 250 and 300 g were used in all experiments. Male rats were used to Accepted for publication Jan 20, 1996. Address reprint requests to Prof Yacoub, National Heart & Lung Institute, Heart Science Centre, Harefidd Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom.
© 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1996;61:1407-12)
Experimental Preparation The isolated, working rat heart preparation, which has been described in detail elsewhere [11], was used in this study. Briefly, in this left heart preparation, oxygenated Krebs-Henseleit bicarbonate buffer (NaCI, 118.5 mmol/L; NaHCO3, 25 mmol/L; KC1, 4.75 mmol/L; MgSO 4, 1.19 mmol/L; KH2PO 4, 1.18 mmol/L; CaC12, 2.5 mmol/L), pH 7.4, containing glucose (11.1 mmol/L) and gassed with 95% oxygen and 5% carbon dioxide at 37°C, enters the cannulated left atrium and passes into the left ventricle, from which it is spontaneously ejected through an aortic cannula against a hydrostatic pressure of 100 cm H20. The heart continues to eject as long as the pressure generated in the left ventricle is greater than 100 cm H20. Total cardiopulmonary bypass with maintained coronary 0003-4975/96/$15.00 PII S0003-4975(96)00085-9
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A M R A N I ET A L KINETICS OF INDUCTION OF HEAT-SHOCK PROTEINS
Ann Thorac Surg 1996;61:1407-12
Table 1. Relation Between Amount of Heat-Shock Protein Induced at Different Intervals and Postischemic Recoverya-c Time (h)
HSP 70 (arbitrary units)
CO
PAP
CF
EF
0.08 + 0.05 0.14 -+ 0.06 0.10 + 0.09 0.18 + 0.04 0.35 + 0.09 0.65 _+ 0.10 0.62 -+ 0.05 0.52 ± 0.04 0.34 + 0.02 0.24 -+ 0.05 0.10 -+ 0.03
51.9 +- 5.0 58.1 -+ 2.9 55.9 -+ 3.9 50.8 + 4.0 57.3 + 1.9 74.0 ± 2.4 70.8 -+ 3.8 78.3 ± 4.0 49.9 ± 5.9 52.8 -+ 3.0 55.9 -+ 3.3
60.9 -+ 5.0 62.1 + 4.0 68.4 -+ 3.9 65.5 ~+2.7 60.2 -+ 4.3 79.3 -+ 5.0 75.1 -+ 2.2 81.0 -~ 3.5 65.5 + 4.5 59.8 ± 5.6 68.0 + 3.0
78.9 -+ 3.0 71.8 -+ 3.3 82.2 -+ 1.7 75.5 -+ 2.5 79.3 + 2.0 89.8 -+ 1.9 95.0 -+ 2.2 92.3 -+ 2.7 83.0 -+ 1.2 75.5 ± 4.3 80.0 -+ 1.0
18.9 + 8.0 22.8 + 7.2 20.7 -+ 5.9 26.3 -+ 6.6 24.9 -+ 6.9 58.3 -+ 7.2 52.0 -+ 6.3 47.7 ± 5.1 25.7 + 3.3 19.9 -+ 4.9 28.3 + 5.7
0 0
57.0 ± 2.8 53.9 + 1.9
63.4 -~ 3.1 60.7 ~ 4.7
76.6 ± 3.6 71.7 ± 3.7
20.3 _+3.8 31.0 ± 6.4
12 14 16 18 20 24 26 30 36 48 96 Control d Sham a
a Each v a l u e r e p r e s e n t s t h e m e a n ± t h e s t a n d a r d e r r o r of t h e m e a n o f six h e a r t s , b P o s t i s c h e m i c r e c o v e r y d a t a a r e s h o w n as p e r c e n t a g e of p r e i s c h e m i c value, c S i g n i f i c a n t d i f f e r e n c e s are s h o w n in F i g u r e 1. a T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n control a n i m a l s a n d t h o s e h a v i n g s h a m procedures. CF - c o r o n a r y flow;
CO = cardiac output;
EF = e n d o t h e l i a l function;
perfusion can be simulated by clamping the left atrial cannula and introducing perfusion fluid at 37°C from a reservoir 100 cm above the heart. Several variables, including cardiac output (CO), peak aortic pressure (PAP), and coronary flow (CF), are measured to reflect cardiac function. The most representative m e a s u r e m e n t of mechanical function is CO. Using this preparation, which is essentially that described by Langendorff, the heart continues to beat but does not p er f o r m external work. Ischemic cardiac arrest can be p r o d u c e d by clamping the aortic cannula. At this time, a cardioplegic solution is infused into a side-arm of the aortic cannula. During the ischemic period, the heart is maintained u n d e r h y p o t h e r m i a (4°C) by a cooling circuit. St. T h o m a s ' Hospital cardioplegic solution No. 1, supplied as concentrate (David Bull Laboratories, Mulgrave, Victoria, Australia), was diluted in a Ringer's solution (Travenol Laboratories, Thetford, Norfolk, England) and passed t h r o u g h an 0.2-p,m filter (Pall Biomedical, Glen Cove, NY).
Endothelial Function Endothelial function was assessed through observations of preischemic and postischemic coronary flow responses to 5-hydroxytryptamine (5-HT). This vasodilatory response is e n d o t h e l i u m d e p e n d e n t . In the intact e n d o t h e lium, 5-HT causes vasodilation t h r o u g h the release of e n d o t h e l i u m - d e r i v e d relaxing factor, w h e r e a s in the presence of endothelial damage, it causes vasoconstriction by a direct effect on smooth muscle. O u r protocol for this test was described in earlier studies [12]. After excision of the heart and aortic cannulation, Langendorff perfusion was initiated at 37°C. Coronary flow was m o n i t o r e d by an in-line electromagnetic flow probe (20-mL ECM2; Scalar, Delft, the Netherlands) proximal to the aortic cannula and c o n n e c t e d to its compatible fl o wm e te r (MDL 1401; Scalar). This p r o v i d e d
H S P 70 - h e a t - s h o c k p r o t e i n 70;
P A P = p e a k aortic p r e s s u r e .
an accurate (0.0 to 40.0 m L/ m i n ) digital r ead o u t of m e a n CF and a simultaneous hard-copy recording through a connection with a chart recorder (series 3000; G oul d Electronics, Hainhault, Essex, England), which allowed accurate m o n i t o r i n g of steady-state conditions (less than 0.3 m L / m i n change in CF over 3 minutes). After 9 to 13 minutes, the initial baseline CF was recorded. The Langendorff infusion was switched to one containing an additional 10 -5 mol/L 5-HT (Sigma C h e m ical Co, Poole, Dorset, England). The ensuing vasodilator response was monitored, and w h e n the steady state had b e e n r each ed (between 5 and 7 minutes), coronary flow was recorded. After this period, 5-HT was w a s h e d out by switching back to ordinary Krebs-Henseleit solution until a steady state had b e e n reached (between 5 and 7 minutes). The heart was then subjected to a 10-mL h y p o t h e r m i c (4°C) infusion with the cardioplegic solution and m a i n t a i n e d i m m e r s e d in the same solution for 4 hours at 4°C. At the end of the ischemic period, the heart was r e p e r f u s e d in the Langendorff m o d e at 37°C for at least 15 minutes. W h e n the baseline CF had b e e n reestablished, the heart was again subjected to the same protocol of sequential infusion of 5-HT and KrebsHenseleit solution as in the preischemic period.
Assessment of Heat-Shock Protein Expression The induction of HSP 70 was assessed by SDS PAGE and W est er n i m m u n o b l o t t i n g as previously described [4, 9]. Proteins of w h o l e - h e a r t h o m o g e n a t e s solubilized in 3% wt/vol SDS were separated on 10% SDS gels. We s t e r n blots on nitrocellulose m e m b r a n e s were p r o b e d with an antibody specific to inducible HSP 70 (Amersham). The result was visualized with peroxidase-conjugated secondary antibody and e n h a n c e d chemiluminescence. Hyperfilm MP was exposed to blots treated with e n h a n c e d c h e m i l u m i n e s c e n c e for 30 seconds and d e v e l o p e d in an automatic film processor. After e n h a n c e d c h e m i l u m i n e s -
A n n Thorac S u r g 1996;61:1407-12
AMRANI ET AL KINETICS OF INDUCTION OF H E A T - S H O C K PROTEINS
b
~o.8 'E o.7
~
0.6
m 0.5
~ 03 a. 0.1 24
48
72
Time interval (hours) a
b
c;
, ~" E ~ T • 3 40
~
T-~
5 =3o~.
/ /
~
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/ Response to 5-HT/
~i---____-
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Time interval (hours) b
a
I
I
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72
96
1409
14, 16, 18, 20, 24, 30, 36, 48, a n d 96 hours after heat shock. In the control group, no p r o c e d u r e was performed. In the s h a m - o p e r a t i o n group, animals were anesthetized without heat stress, allowed to recover for 24 hours, a n d then sacrificed. The femoral vein was exposed, a n d h e p a r i n s o d i u m (200 IU) was injected. O n e m i n u t e later, the h e a r t was excised a n d i m m e d i a t e l y placed into cold (10°C) perfusion fluid. The aorta was cannulated, a n d Langendorff perfusion was initiated for a 3-minute w a s h o u t period. Endothelial function studies were p e r f o r m e d in the Langendorff m o d e (nonworking). Hearts d e s i g n a t e d to assess mechanical function were converted to a working m o d e p r e p a r a t i o n for 20 minutes, d u r i n g which control values for aortic a n d coronary flow rates, CO (sum of aortic a n d coronary flows), a n d PAP were recorded. A n y h e a r t that did not reach a steady level of function with a stable heart rate a n d a CO greater than 65 m L / m i n was rejected. Two hearts were rejected, one because of a p r o l o n g e d ischemic time in m o u n t i n g it on the Langendorff a p p a r a t u s and one because of a leak from incorrect manipulation. At the e n d of the control period, the atrial a n d aortic cannulas were clamped, a n d the heart was i m m e d i a t e l y subjected to a coronary infusion of 10 mL of h y p o t h e r m i c (4°C) cardioplegic solution a n d m a i n t a i n e d i m m e r s e d in a state of h y p o t h e r m i c arrest for 4 hours. At the e n d of the ischemic arrest period, each heart was r e p e r f u s e d at 37°C in the Langendorff m o d e for 15 minutes a n d then converted to a 20-minute p e r i o d of working m o d e at the end of which the recovery values of aortic a n d coronary flow rates a n d PAP were recorded.
Time interval (hours)
Expression Fig 1. Relation between level of heat-shock protein 70 (HSP 70) induced at different intervals and postischemic recovery of cardiac output (CO), peak aortic pressure (PAP), and coronary flow as well as vasodilatory effect of 5-hydroxytryptamine (5-HT), an index of endothelial function. Each point represents the mean of six hearts (+_ the standard error of the mean). Significant differences were as follows: a versus b and b versus c, both p < 0.05; a versus c, p = not significant; between values obtained within a, b, and c, p = not significant. (a = values obtained before 24 hours; b = values obtained at 24, 26, and 30 hours; c = values obtained after 30 hours; C = control.)
of Results
The postischemic recovery values of mechanical function a n d endothelial function were expressed as a percentage of the preischemic values. The level of HSP 70 was expressed in arbitrary units. An analysis of variance was p e r f o r m e d with Scheff6's correction factor. The significance of differences b e t w e e n groups was d e t e r m i n e d with a n o n p a i r e d S t u d e n t ' s t test, a n d significance was a s s u m e d w h e n the p value was 0.05 or less. Values are given as the m e a n ± the s t a n d a r d error of the mean. Results
cence, antibodies were r e m o v e d from blots b y incubation in a solution of 2% wt/vol SDS, 6.25% vol/vol 1 mol/L tris-HCL, p H 6.8, a n d 0.7% vol/vol 2-mercaptoethanol. Proteins were then visualized b y staining with 0.01% a m i d o black in a solution of m e t h a n o l water, a n d acetic acid (45:45:10 vol/vol). A m i d o b l a c k - s t a i n e d blots a n d e n h a n c e d - c h e m i l u m i n e s c e n c e films were s c a n n e d using a Molecular Dynamics 300A laser densitometer, a n d HSP 70 levels were d e t e r m i n e d as a proportion of total protein l o a d e d using the PDQUEST software package (PDL H u n t ington, NY). Experimental
Time Course
A n i m a l s were anesthetized with halothane mixed with 95% oxygen plus 5% carbon dioxide a n d sacrificed at 12,
Mechanical
and Endothelial
Function
In the control group (n = 6), postischemic recovery (mean percentage of preischemic value) of CO, PAP, a n d CF was 57.0 + 2.8, 63.4 _+ 3.1, a n d 76.6 --_ 3.6, respectively. Postischemic recovery of the coronary r e s p o n s e to 5-HT (mean percentage of preischemic value + s t a n d a r d error of the mean) was 20.3 -+ 3.8. Twenty-four hours after heat shock, postischemic recovery of CO, PAP, CF, a n d coronary flow response to 5-HT (as percentage of preischemic value ± s t a n d a r d error of the mean) was 74.0 ± 2.4, 79.3 ± 5.0, 89.8 ± 1.9, a n d 58.3 ± 7.2, respectively. Similar values (p = not significant) were seen at 26 a n d 30 hours after heat shock. Values o b t a i n e d at 12, 14, 16, 18, a n d 20 hours after heat stress were similar to those in the control a n d s h a m -
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A M R A N I ET A L KINETICS OF INDUCTION OF HEAT-SHOCK PROTEINS
Ann Thorac Surg 1996;61:1407-12
Table 2. Preischemic and Postischemic Mechanical and Endothelial Function at Different Intervals After Heat Stress
Time (h)
CO (mL/min) Pre
PAP (cm H20) Post
12 14 16 18 20 24 26 30 36 48 96
87.5 80.5 74.1 90.3 78.6 77.3 81.3 79.3 69.8 79.3 82.4
± 3.5 + 5.5 ± 5.3 _+ 4.9 _+ 8.2 ± 5.8 ± 7.2 + 9.9 + 5.4 ± 6.6 _+ 8.2
Control Sham
88.0 _+ 5.5 80.6 + 4.7
45.4 46.7 41.4 52.1 45.0 57.2 57.6 62.3 34.2 41.8 46.0
-- 5.4 ± 4.5 ± 7.2 ± 6.3 ± 6.6 ± 4.4 ± 5.1 _+ 5.3 ± 3.8 - 6.3 _+ 6.2
50.2 ÷ 7.0 43.4 ± 6.0
Pre 182.1 184.9 178.2 194.1 174.0 172.3 188.5 196.5 188.8 189.0 173.0
± 8.8 ± 9.1 ± 9.5 + 7.9 ± 6.0 ± 4.9 _+ 6.2 + 9.3 ± 6.5 + 8.9 + 4.9
180.0 ± 8.0 182.0 ± 8.3
CF (mL/min) Post
110.8 114.3 121.0 127.1 104.5 135.8 141.4 158.3 123.6 113.3 117.5
Pre
+ 8.5 ± 6.4 -+ 8.0 + 5.8 ± 8.8 + 5.0 ± 6.2 + 5.9 ± 7.1 _+ 3.9 _+ 5.0
14.7 15.0 13.0 12.7 15.5 13.3 15.4 16.0 12.8 14.7 15.5
117.3 ± 5.0 109.2 ± 7.9
+ 0.9 ± 1.1 _+ 0.5 + 0.7 ± 1.0 + 0.7 _+ 0.9 ± 0.7 _+ 0.9 _+ 0.6 ± 0.5
16.3 + 0.5 15.7 + 0.7
EF (% increase of CF) Post
11.6 10.7 10.7 9.6 12.3 11.0 14.6 14.7 10.6 11.1 12.1
± 0.6 _+ 0.8 -+ 0.6 -+ 0.8 ± 0.3 + 0.4 + 0.7 ± 0.8 ± 0.8 + 0.4 + 0.2
12.5 ± 0.8 12.2 -+ 0.3
Pre 65.5 72.0 57.0 63.5 70.0 59.5 75.0 69.0 74.0 68.8 70.0
± 5.5 _+ 7.0 _+ 5.7 +_ 3.3 ± 9.7 + 5.7 _+ 6.6 ± 8.0 _+ 5.6 _+ 9.3 ± 7.1
74.0 ± 4.7 63.5 ± 7.0
Post 12.2 16.0 11.7 16.5 16.8 34.2 39.0 32.3 18.5 13.5 19.6
± 2.9 ± 5.5 + 4.4 - 5.2 ± 7.0 ± 6.2 + 5.1 _+ 4.9 + 3.9 ± 7.7 _+ 4.1
14.8 ± 7.1 19.8 ± 6.2
a Each v a l u e r e p r e s e n t s t h e m e a n _+ t h e s t a n d a r d e r r o r of t h e m e a n of six hearts. CF - c o r o n a r y flow;
C O = c a r d i a c output;
EF = e n d o t h e l i a l function;
o p e r a t i o n g r o u p s a n d significantly l o w e r t h a n t h o s e obt a i n e d at 24, 26, a n d 30 h o u r s (p < 0.05). Likewise, v a l u e s o b t a i n e d after 30 h o u r s w e r e c o m p a r a b l e to t h o s e in the control a n d s h a m - o p e r a t i o n g r o u p s a n d significantly l o w e r t h a n t h o s e o b t a i n e d at 24, 26, a n d 30 h o u r s (p 0.05). Table 1 a n d F i g u r e 1 s h o w the c o r r e s p o n d i n g p o s t i s c h e m i c r e c o v e r i e s to the t i m e intervals s t u d i e d . Significance is r e p o r t e d in Figure 1. Table 2 s h o w s t h e p r e i s c h e m i c a n d p o s t i s c h e m i c v a l u e s of CO, PAP, CF, a n d c o r o n a r y flow r e s p o n s e to 5-HT.
H S P 70 N o i n d u c t i o n of H S P 70 w a s d e t e c t a b l e in a n i m a l s in the control a n d s h a m - o p e r a t i o n g r o u p s . H e a t - s t r e s s p r o t e i n 70 was d e t e c t a b l e in h e a t - s h o c k e d a n i m a l s f r o m 12 h o u r s after h e a t t r e a t m e n t . P e a k H S P levels w e r e o b s e r v e d at 24 h o u r s a n d d e c l i n e d r a p i d l y after 30 hours. T h e H S P 70 levels, e x p r e s s e d as a p r o p o r t i o n of the total p r o t e i n l o a d e d for e a c h s a m p l e ( m e a s u r e d on a m i d o - s t a i n e d blots), are s h o w n in Table 1. Comment T h e p r e s e n t s t u d y has s h o w n that after p r o l o n g e d cardioplegic arrest, the p o s t i s c h e m i c r e c o v e r y of b o t h cardiac m e c h a n i c a l f u n c t i o n a n d e n d o t h e l i a l f u n c t i o n was significantly i m p r o v e d w h e n t h e i n t e r v a l b e t w e e n h e a t stress a n d i s c h e m i a r a n g e d f r o m 24 to 30 hours. This i n t e r v a l c o r r e l a t e d w i t h the m a x i m a l h e a t - s h o c k res p o n s e as d e t e r m i n e d f r o m levels of the i n d u c i b l e stress p r o t e i n H S P 70. W h e n any cell is s u b j e c t e d to s u b l e t h a l h y p e r t h e r m i a , a v a r i e t y of a d a p t i v e m o d i f i c a t i o n s take place t h a t s e r v e to p r o t e c t the cell f r o m s u b s e q u e n t i n c r e a s e s in t e m p e r ature or o t h e r stresses [1-4]. In a d d i t i o n to h y p e r t h e r m i a , o t h e r stresses i n c l u d i n g i s c h e m i c p r e c o n d i t i o n i n g [13], c h e m i c a l a g e n t s [14], a n d p r e s s u r e o v e r l o a d [15] i n d u c e
PAP
p e a k aortic p r e s s u r e .
H S P synthesis. This s u g g e s t s that t h e r e s p o n s e m a y r e p r e s e n t a g e n e r a l i z e d cellular d e f e n s e m e c h a n i s m a n d h e n c e , the c u r r e n t c o n c e p t of " c r o s s t o l e r a n c e " [6]. Bec a u s e it uses an intrinsic cellular d e f e n s e m e c h a n i s m b e f o r e the v e r y o n s e t of i s c h e m i a , this a p p r o a c h app e a r e d to be a n e l e g a n t t h e r a p e u t i c a l t e r n a t i v e to p r o t e c t against cardiac i s c h e m i a [16]. O v e r the last d e c a d e , the p r o t e c t i v e effect of H S P s against i s c h e m i c d a m a g e has b e e n w i d e l y i n v e s t i g a t e d in v a r i o u s e x p e r i m e n t a l m o d e l s a n d has led to p r o m i s i n g results in m o s t studies. H o w e v e r , s o m e i n v e s t i g a t o r s [6, 13, 17] s t r e s s e d the lack of c o r r e l a t i o n b e t w e e n t h e " p r e s e n c e " of H S P s a n d t h e i r p r o t e c t i v e role. This d i s c r e p a n c y s u g g e s t s that a critical a m o u n t of H S P is n e c e s s a r y to elicit a p r o t e c t i v e effect a n d that this "critical v a l u e " is m o r e i m p o r t a n t to d e f i n e t h a n t h e m e r e p r e s e n c e of HSPs. This is f u r t h e r e m p h a s i z e d by t h e possibility that e a c h p a r t i c u l a r e x p e r i m e n t a l m o d e l (animals, t y p e of stress stimuli, l e n g t h of i s c h e m i a , t y p e of p e r f u s i o n ) m i g h t r e s u l t in different "critical p r o t e c t i v e " c o n c e n t r a t i o n s of HSPs. A t e m p o r a l r e l a t i o n s h i p b e t w e e n the a p p e a r a n c e a n d s u b s e q u e n t d i s a p p e a r a n c e of H S P s w i t h f u n c t i o n a l rec o v e r y has b e e n p r e v i o u s l y r e p o r t e d in n o n q u a n t i t a t i v e H S P studies in t h e i s o l a t e d rat h e a r t s u b j e c t e d to l o w flow i s c h e m i a [10] a n d after r e g i o n a l i s c h e m i a in the rabbit h e a r t [7]. In t h e p r e s e n t study, w e a s s e s s e d the h e a t - s h o c k res p o n s e b y m e a s u r i n g H S P 70 at different i n t e r v a l s r a n g ing f r o m 12 to 96 h o u r s after h e a t stress u s i n g a s e m i q u a n t i t a t i v e m e t h o d . In addition, for e a c h interval, w e e v a l u a t e d the p o s t i s c h e m i c r e c o v e r y of m e c h a n i c a l a n d e n d o t h e l i a l f u n c t i o n after p r o l o n g e d c a r d i o p l e g i c arrest. E n d o t h e l i a l f u n c t i o n was s t u d i e d b e c a u s e in p r e v i o u s work, w e [18] h a v e d e m o n s t r a t e d its i n f l u e n c e on m y o cardial contractility. To date, t h e r e are n o c o m p a r a t i v e d a t a r e g a r d i n g i n d u c t i o n of H S P s in the different cellular c o m p o n e n t s . O u r results s t r o n g l y s u g g e s t that H S P 70 is
Ann Thorac Surg 1996;61:1407-12
i n d u c e d in endothelial cells. This r e m a i n s to be validated in isolated cell preparations. We f o u n d that both maximal levels of HSP 70 as well as optimal recovery of mechanical a n d endothelial function were seen at 24, 26, a n d 30 hours. For all other intervals, the concentration of HSP 70 was significantly lower a n d was associated with a recovery similar to the control group (no heat stress). Thus, it appears that a critical a m o u n t of HSP is necessary to afford protection against ischemia. This study has several limitations, which include the fact that although we have defined the relative a m o u n t of HSP 70 in relation to other proteins in the myocardium, we have not defined the absolute a m o u n t of HSP 70 in the cells concerned. In addition, we have chosen to study one type of HSP (HSP 70), the one that is most c o m m o n l y expressed. Other HSPs could have similar or more important influence on myocardial protection. Finally, the clinical relevance of our results is influenced by the fact that the use of heat stress in the clinical setting is not practical. Other strategies for i n d u c i n g HSPs need to be developed. It is h o p e d that the data presented here will be of value in u n d e r s t a n d i n g a n d possibly applying methods of e n h a n c i n g the intrinsic protective mechanisms of the heart against ischemic damage. We thank Mr Jamal A. Amrani for help with the statistical analysis.
References 1. Hightower LE. Heart shock proteins, chaperones, and proteotoxicity. Cell 1991;66:191-7. 2. Li CG, Werb Z. Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblast. Proc Natl Acad Sci USA 1982;79:3218-22. 3. Sanchez Y, Linquist SL, HSP104 is required for induced thermotolerance. Science 1990;248:1112-5. 4. Marber MS, Latchman DS, Walker JM, Yeilon DM. Cardiac stress protein elevation 24 hours after brief ischemic or heat stress is associated with resistance to myocardial infarction. Circulation 1993;88:1264-72. 5. Hutter MM, Sievers RE, Barbosa V, Wolfe CL. Heat-shock
AMRANIET AL KINETICSOF INDUCTIONOF HEAT-SHOCKPROTEINS
6.
7. 8.
9.
10. 11. 12.
13.
14.
15.
16. 17. 18.
1411
proteins induction in rat hearts. A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 1994;89:355-60. Donnelly TJ, Sievers RE, Vissern FL, Welch WJ, Wolfe CL. Heat-shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion? Circulation 1992;85:769-78. Currie RW, Tanguay RM, Kingma JG. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 1993;87:963-71. Amrani M, Corbett J, Allen NJ, et al. Induction of heat-shock proteins enhances myocardial and endothelial functional recovery after prolonged cardioplegic arrest. Ann Thorac Surg 1994;57:157-60. Amrani M, Allen NJ, O'Shea J, et al. Role of catalase and heat-shock protein on recovery of cardiac endothelial and mechanical function after ischemia. Cardioscience 1993;4: 193-8. Karmazyn M, Mailer K, Currie RW. Acquisition and decay of heat-shock proteins enhanced postischemic ventricular recovery. Am J Physiol 1990;259:H424-31. Neely JR, Leibermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat hearts. Am J Physiol 1967;212:804-14. Amrani M, Ledingham S, Jayakumar J, et al. Detrimental effect on the efficacy of the University of Wisconsin solution when used for cardioplegia at moderate hypothermia. Comparison with the St. Thomas Hospital solution at 4°C and 20°C. Circulation 1992;86(Suppl 2):280-8. Tanaka M, Fujiwara H, Yamasaki K, et al. Ischemic preconditioning elevates cardiac stress protein but does not limit infarct size 24 or 48 hours later in rabbits. Am J Physiol 1994;267:H1476-82. Maulik N, Engelman RM, Wei Z, Lu D, Rousou JA, Das DK. Interleukin-1 alpha preconditioning reduces myocardial ischemia reperfusion injury. Circulation 1993;88(Suppl 2): 387-94. Delcayre C, Samuel JL, Marotte F, Best-Belpomme M, Mercadier JJ, Rappaport L. Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J Clin Invest 1988;82:460-8. Liu X, Engelman RM, Moraru H, et al. Heat shock: a new approach for myocardial preservation in cardiac surgery. Circulation 1992;86(Suppl 2):358-63. Yellon DM, Iliodromitis E, Latchman DS, et al. Whole body heat stress fails to limit infarct size in the reperfused heart. Cardiovasc Res 1992;26:342-6. Amrani M, O'Shea J, Allen N, et al. Role of basal release of nitric oxide on coronary flow and mechanical performance in the isolated rat heart. J Physiol (Lond) 1992;456:681-7.
INVITED COMMENTARY W h e n living cells are subjected to a short elevation in temperature, certain proteins, k n o w n as heat-stress or heat-shock proteins (HSPs), are synthesized. These proteins have b e e n shown to s u b s e q u e n t l y protect the myocyte from not only further heat stress, b u t other metabolic insults, including ischemia/reperfusion injury [1, 2]. The induction of these HSPs has b e e n shown to protect the m y o c a r d i u m from ischemic damage in a n u m b e r of different models a n d species. The effects have i n c l u d e d a reduction in infarct size as d e m o n s t r a t e d by tetrazolium staining, reduced release of creatine kinase, a n d imp r o v e m e n t in contractile functions. Increased levels of HSP have b e e n associated with preservation of tissue adenosine triphosphate after ischemia-reperfusion a n d
decreased production of oxygen-derived free radicals after reperfusion [3]. However, whether one or a combination of these m e c h a n i s m s is mediated through HSP resulting in myocardial protection r e m a i n s u n k n o w n . In the study, A m r a n i a n d associates investigated postischernic recovery of mechanical a n d endothelial cell function in a model of hypothermic cardioplegic arrest a n d correlated this with the synthesis of inducible form of HSP 70. Their results demonstrate that maximal levels of HSP 70 correlated with postischernic recovery observed at 24, 26, a n d 30 hours after hypothermic cardioplegic arrest. For other time intervals, the postischemic functional recovery was comparable with control, although the levels of HSP 70 were still detectable.
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AMRANI ET AL KINETICS OF I N D U C T I O N OF H E A T - S H O C K PROTEINS
This study raises a very important question: what is the critical threshold of HSP 70 to induce protection in the heart? Although the cellular levels of HSP 70 were significantly increased at several time intervals (18, 20, 36, and 48 hours after heat shock), apparently the level of protein was not high enough to protect the myocardium against hypothermic cardioplegic arrest. The results of this study differ somewhat from other studies. For example, Currie and associates [1] reported increased expression of HSP 71 correlated with cardioprotection at 24 hours but not 40 hours after heat shock. A preliminary study by Shipley and colleagues [4] demonstrated rapid induction and accumulation of both HSP 27 and HSP 72 four hours after heat stress. Interestingly, no myocardial protection was afforded until 24 hours after heat stress, and protection was gone by 30 hours after heat stress. The synthesis of HSP after heat stress was rapid, reaching more than 80% of maximum within 4 hours of initial insult. These results suggested that the myocardial protection afforded by heat stress cannot be solely explained on the basis of HSP expression, and may be dependent on posttranslational modification of translocation of HSP, or may be dependent on other as yet unidentified factors. Clearly, there is considerable controversy in this important area, and rigorous studies are required to further elucidate the mechanisms of heat stress induced cardiac protection. Nevertheless, Amrani and colleagues deserve compliments for investigating the role of HSP 70 in this unique model of hypothermic cardioplegic arrest. To borrow a term from our high-tech colleagues, if cardioplegia represented the first generation of cardiac protection, preconditioning represents the second generation. Obviously heat shock, being nonphysiologic, is only a model,
A n n Thorac S u r g 1996;61:1407-12
but the race is on in the laboratory on two fronts: first, the development of clinically applicable preconditioning either metabolically or pharmacologically, and second, the isolation and characterization of these unique HSPs. The prolonged ischemic time associated with cardiac transplantation appears to be a logical initial problem to begin this second generation approach. If a direct cause and effect relationship could be established for expression of HSP 70 versus protection of ischemic myocardium, the protein will open the door to new therapeutic options to reduce damage caused by ischemic heart disease of multiple etiologies.
Rakesh C. Kukreja, PhD Michael L. Hess, MD Eric Lipman Laboratories of Molecular and Cellular Cardiology Division of Cardiology Medical College of Virginia Richmond, VA 23298 References 1. Currie RW, Tanguay RM, Kingma JG Jr. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 1993;87:963--71. 2. Karmazyn M, Mailer K, Currie RW. Acquisition and decay of heat-shock enhanced postischemic ventricular recovery. Am J Physiol 1990;259:H424-31. 3. Mocanu MM, Steare SE, Evans MC, Nugent JH, Yellon DM. Heat stress attenuates free radical release in the isolated perfused rat heart. Free Radic Biol Med 1993;15:459-63. 4. Shipley JB, Qian Y-Z, Levasseur JE, Kukreja RC. Expression of the stress proteins HSP-27 and HSP-72 in rat heart does not correlate with ischemic tolerance after heat shock. Circulation 1995;92(Suppl 1):654.
Amrani,
M i c h a e l J. D u n n ,
MD, PhD, Joseph
PhD, and Magdi
Corbett, PhD, Samuel
Y. B o a t e n g , BSc,
H. Yacoub, FRCS
National Heart & Lung Institute, Heart Science Centre, Harefield Hospital, Harefield, Middlesex, United Kingdom
Background. Heat-shock proteins are known to enhance cardiac resistance to ischemia. Methods. To evaluate the kinetics of heat-shock protein 70 in relation to its effect on postischemic recovery of cardiac mechanical (cardiac output) and endothelial function (as percentage increase of coronary flow in response to 5-hydroxytryptamine), isolated rat hearts were subjected to prolonged hypothermic cardioplegic arrest at different intervals ranging from 12 to 96 hours after heat stress (n = 6 in each interval). Results. Immunoblotting showed the maximal level of heat-shock protein 70, 0.65 - 0.10 (arbitrary units + standard error of the mean), at 24 hours after heat shock
and similar values at 26 and 30 hours (p = not significant). Postischemic recovery of cardiac output and endothelial function (percentage of preischemic value -+ standard error of the mean) observed at 24 hours was 74.0 +-2.4 and 58.3 -+ 7.2, respectively. Similar values were observed at 26 and 30 hours (p = not significant). Conclusions. In a protocol mimicking conditions for cardiac transplantation, postischemic recovery of cardiac output and endothelial function was improved when the interval between heat stress and ischemia ranged from 24 to 30 hours. This correlated with an apparently critical amount of heat-shock protein 70.
eat-shock proteins (HSPs) belong to a large group of proteins with a wide range of molecular weights. Some HSPs are constitutively expressed, whereas others are induced by a variety of mechanisms but are preferentially expressed after heat shock [1-4]. Heat-shock proteins are known to protect the cell against a further stress. Of particular interest is the finding that HSPs enhance cardiac resistance to ischemia. This has been demonstrated in numerous experimental models of low-flow or regional ischemia using different stress stimuli [4-7]. We ]8, 9] have previously shown a protective effect of heat stress on both endothelial function and mechanical function after cardioplegic arrest. However, the exact time course of induction and the relation between the amount of HSP induced and the protective effect has not been adequately investigated, particularly after cardioplegic arrest [6, 7, 10]. The aim of the present study was to determine the levels of HSP 70 induction at various intervals after heat stress and to identify the amount of HSP affording the maximal recovery of endothelial and mechanical function in a protocol mimicking conditions for cardiac transplantation.
avoid a potential lack of homogeneity related to hormonal cycle. Six hearts were studied in each group. In all studies, animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985). Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and then placed on a temperature-controlled heating pad (IMS K-Temp control unit; Concleton, Cheshire, England) set at 45°C until body temperature reached 42°C. Body temperature was monitored with a rectal temperature probe and maintained between 42° and 42.5°C for 15 minutes as previously described [10].
H
Material and M e t h o d s Sprague-Dawley rats weighing between 250 and 300 g were used in all experiments. Male rats were used to Accepted for publication Jan 20, 1996. Address reprint requests to Prof Yacoub, National Heart & Lung Institute, Heart Science Centre, Harefidd Hospital, Harefield, Middlesex, UB9 6JH, United Kingdom.
© 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 1996;61:1407-12)
Experimental Preparation The isolated, working rat heart preparation, which has been described in detail elsewhere [11], was used in this study. Briefly, in this left heart preparation, oxygenated Krebs-Henseleit bicarbonate buffer (NaCI, 118.5 mmol/L; NaHCO3, 25 mmol/L; KC1, 4.75 mmol/L; MgSO 4, 1.19 mmol/L; KH2PO 4, 1.18 mmol/L; CaC12, 2.5 mmol/L), pH 7.4, containing glucose (11.1 mmol/L) and gassed with 95% oxygen and 5% carbon dioxide at 37°C, enters the cannulated left atrium and passes into the left ventricle, from which it is spontaneously ejected through an aortic cannula against a hydrostatic pressure of 100 cm H20. The heart continues to eject as long as the pressure generated in the left ventricle is greater than 100 cm H20. Total cardiopulmonary bypass with maintained coronary 0003-4975/96/$15.00 PII S0003-4975(96)00085-9
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A M R A N I ET A L KINETICS OF INDUCTION OF HEAT-SHOCK PROTEINS
Ann Thorac Surg 1996;61:1407-12
Table 1. Relation Between Amount of Heat-Shock Protein Induced at Different Intervals and Postischemic Recoverya-c Time (h)
HSP 70 (arbitrary units)
CO
PAP
CF
EF
0.08 + 0.05 0.14 -+ 0.06 0.10 + 0.09 0.18 + 0.04 0.35 + 0.09 0.65 _+ 0.10 0.62 -+ 0.05 0.52 ± 0.04 0.34 + 0.02 0.24 -+ 0.05 0.10 -+ 0.03
51.9 +- 5.0 58.1 -+ 2.9 55.9 -+ 3.9 50.8 + 4.0 57.3 + 1.9 74.0 ± 2.4 70.8 -+ 3.8 78.3 ± 4.0 49.9 ± 5.9 52.8 -+ 3.0 55.9 -+ 3.3
60.9 -+ 5.0 62.1 + 4.0 68.4 -+ 3.9 65.5 ~+2.7 60.2 -+ 4.3 79.3 -+ 5.0 75.1 -+ 2.2 81.0 -~ 3.5 65.5 + 4.5 59.8 ± 5.6 68.0 + 3.0
78.9 -+ 3.0 71.8 -+ 3.3 82.2 -+ 1.7 75.5 -+ 2.5 79.3 + 2.0 89.8 -+ 1.9 95.0 -+ 2.2 92.3 -+ 2.7 83.0 -+ 1.2 75.5 ± 4.3 80.0 -+ 1.0
18.9 + 8.0 22.8 + 7.2 20.7 -+ 5.9 26.3 -+ 6.6 24.9 -+ 6.9 58.3 -+ 7.2 52.0 -+ 6.3 47.7 ± 5.1 25.7 + 3.3 19.9 -+ 4.9 28.3 + 5.7
0 0
57.0 ± 2.8 53.9 + 1.9
63.4 -~ 3.1 60.7 ~ 4.7
76.6 ± 3.6 71.7 ± 3.7
20.3 _+3.8 31.0 ± 6.4
12 14 16 18 20 24 26 30 36 48 96 Control d Sham a
a Each v a l u e r e p r e s e n t s t h e m e a n ± t h e s t a n d a r d e r r o r of t h e m e a n o f six h e a r t s , b P o s t i s c h e m i c r e c o v e r y d a t a a r e s h o w n as p e r c e n t a g e of p r e i s c h e m i c value, c S i g n i f i c a n t d i f f e r e n c e s are s h o w n in F i g u r e 1. a T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e n control a n i m a l s a n d t h o s e h a v i n g s h a m procedures. CF - c o r o n a r y flow;
CO = cardiac output;
EF = e n d o t h e l i a l function;
perfusion can be simulated by clamping the left atrial cannula and introducing perfusion fluid at 37°C from a reservoir 100 cm above the heart. Several variables, including cardiac output (CO), peak aortic pressure (PAP), and coronary flow (CF), are measured to reflect cardiac function. The most representative m e a s u r e m e n t of mechanical function is CO. Using this preparation, which is essentially that described by Langendorff, the heart continues to beat but does not p er f o r m external work. Ischemic cardiac arrest can be p r o d u c e d by clamping the aortic cannula. At this time, a cardioplegic solution is infused into a side-arm of the aortic cannula. During the ischemic period, the heart is maintained u n d e r h y p o t h e r m i a (4°C) by a cooling circuit. St. T h o m a s ' Hospital cardioplegic solution No. 1, supplied as concentrate (David Bull Laboratories, Mulgrave, Victoria, Australia), was diluted in a Ringer's solution (Travenol Laboratories, Thetford, Norfolk, England) and passed t h r o u g h an 0.2-p,m filter (Pall Biomedical, Glen Cove, NY).
Endothelial Function Endothelial function was assessed through observations of preischemic and postischemic coronary flow responses to 5-hydroxytryptamine (5-HT). This vasodilatory response is e n d o t h e l i u m d e p e n d e n t . In the intact e n d o t h e lium, 5-HT causes vasodilation t h r o u g h the release of e n d o t h e l i u m - d e r i v e d relaxing factor, w h e r e a s in the presence of endothelial damage, it causes vasoconstriction by a direct effect on smooth muscle. O u r protocol for this test was described in earlier studies [12]. After excision of the heart and aortic cannulation, Langendorff perfusion was initiated at 37°C. Coronary flow was m o n i t o r e d by an in-line electromagnetic flow probe (20-mL ECM2; Scalar, Delft, the Netherlands) proximal to the aortic cannula and c o n n e c t e d to its compatible fl o wm e te r (MDL 1401; Scalar). This p r o v i d e d
H S P 70 - h e a t - s h o c k p r o t e i n 70;
P A P = p e a k aortic p r e s s u r e .
an accurate (0.0 to 40.0 m L/ m i n ) digital r ead o u t of m e a n CF and a simultaneous hard-copy recording through a connection with a chart recorder (series 3000; G oul d Electronics, Hainhault, Essex, England), which allowed accurate m o n i t o r i n g of steady-state conditions (less than 0.3 m L / m i n change in CF over 3 minutes). After 9 to 13 minutes, the initial baseline CF was recorded. The Langendorff infusion was switched to one containing an additional 10 -5 mol/L 5-HT (Sigma C h e m ical Co, Poole, Dorset, England). The ensuing vasodilator response was monitored, and w h e n the steady state had b e e n r each ed (between 5 and 7 minutes), coronary flow was recorded. After this period, 5-HT was w a s h e d out by switching back to ordinary Krebs-Henseleit solution until a steady state had b e e n reached (between 5 and 7 minutes). The heart was then subjected to a 10-mL h y p o t h e r m i c (4°C) infusion with the cardioplegic solution and m a i n t a i n e d i m m e r s e d in the same solution for 4 hours at 4°C. At the end of the ischemic period, the heart was r e p e r f u s e d in the Langendorff m o d e at 37°C for at least 15 minutes. W h e n the baseline CF had b e e n reestablished, the heart was again subjected to the same protocol of sequential infusion of 5-HT and KrebsHenseleit solution as in the preischemic period.
Assessment of Heat-Shock Protein Expression The induction of HSP 70 was assessed by SDS PAGE and W est er n i m m u n o b l o t t i n g as previously described [4, 9]. Proteins of w h o l e - h e a r t h o m o g e n a t e s solubilized in 3% wt/vol SDS were separated on 10% SDS gels. We s t e r n blots on nitrocellulose m e m b r a n e s were p r o b e d with an antibody specific to inducible HSP 70 (Amersham). The result was visualized with peroxidase-conjugated secondary antibody and e n h a n c e d chemiluminescence. Hyperfilm MP was exposed to blots treated with e n h a n c e d c h e m i l u m i n e s c e n c e for 30 seconds and d e v e l o p e d in an automatic film processor. After e n h a n c e d c h e m i l u m i n e s -
A n n Thorac S u r g 1996;61:1407-12
AMRANI ET AL KINETICS OF INDUCTION OF H E A T - S H O C K PROTEINS
b
~o.8 'E o.7
~
0.6
m 0.5
~ 03 a. 0.1 24
48
72
Time interval (hours) a
b
c;
, ~" E ~ T • 3 40
~
T-~
5 =3o~.
/ /
~
0
\
I
~,
+
/ Response to 5-HT/
~i---____-
24
48
72
g6
Time interval (hours) b
a
I
I
c
rI
1
8 0
24
48
72
96
1409
14, 16, 18, 20, 24, 30, 36, 48, a n d 96 hours after heat shock. In the control group, no p r o c e d u r e was performed. In the s h a m - o p e r a t i o n group, animals were anesthetized without heat stress, allowed to recover for 24 hours, a n d then sacrificed. The femoral vein was exposed, a n d h e p a r i n s o d i u m (200 IU) was injected. O n e m i n u t e later, the h e a r t was excised a n d i m m e d i a t e l y placed into cold (10°C) perfusion fluid. The aorta was cannulated, a n d Langendorff perfusion was initiated for a 3-minute w a s h o u t period. Endothelial function studies were p e r f o r m e d in the Langendorff m o d e (nonworking). Hearts d e s i g n a t e d to assess mechanical function were converted to a working m o d e p r e p a r a t i o n for 20 minutes, d u r i n g which control values for aortic a n d coronary flow rates, CO (sum of aortic a n d coronary flows), a n d PAP were recorded. A n y h e a r t that did not reach a steady level of function with a stable heart rate a n d a CO greater than 65 m L / m i n was rejected. Two hearts were rejected, one because of a p r o l o n g e d ischemic time in m o u n t i n g it on the Langendorff a p p a r a t u s and one because of a leak from incorrect manipulation. At the e n d of the control period, the atrial a n d aortic cannulas were clamped, a n d the heart was i m m e d i a t e l y subjected to a coronary infusion of 10 mL of h y p o t h e r m i c (4°C) cardioplegic solution a n d m a i n t a i n e d i m m e r s e d in a state of h y p o t h e r m i c arrest for 4 hours. At the e n d of the ischemic arrest period, each heart was r e p e r f u s e d at 37°C in the Langendorff m o d e for 15 minutes a n d then converted to a 20-minute p e r i o d of working m o d e at the end of which the recovery values of aortic a n d coronary flow rates a n d PAP were recorded.
Time interval (hours)
Expression Fig 1. Relation between level of heat-shock protein 70 (HSP 70) induced at different intervals and postischemic recovery of cardiac output (CO), peak aortic pressure (PAP), and coronary flow as well as vasodilatory effect of 5-hydroxytryptamine (5-HT), an index of endothelial function. Each point represents the mean of six hearts (+_ the standard error of the mean). Significant differences were as follows: a versus b and b versus c, both p < 0.05; a versus c, p = not significant; between values obtained within a, b, and c, p = not significant. (a = values obtained before 24 hours; b = values obtained at 24, 26, and 30 hours; c = values obtained after 30 hours; C = control.)
of Results
The postischemic recovery values of mechanical function a n d endothelial function were expressed as a percentage of the preischemic values. The level of HSP 70 was expressed in arbitrary units. An analysis of variance was p e r f o r m e d with Scheff6's correction factor. The significance of differences b e t w e e n groups was d e t e r m i n e d with a n o n p a i r e d S t u d e n t ' s t test, a n d significance was a s s u m e d w h e n the p value was 0.05 or less. Values are given as the m e a n ± the s t a n d a r d error of the mean. Results
cence, antibodies were r e m o v e d from blots b y incubation in a solution of 2% wt/vol SDS, 6.25% vol/vol 1 mol/L tris-HCL, p H 6.8, a n d 0.7% vol/vol 2-mercaptoethanol. Proteins were then visualized b y staining with 0.01% a m i d o black in a solution of m e t h a n o l water, a n d acetic acid (45:45:10 vol/vol). A m i d o b l a c k - s t a i n e d blots a n d e n h a n c e d - c h e m i l u m i n e s c e n c e films were s c a n n e d using a Molecular Dynamics 300A laser densitometer, a n d HSP 70 levels were d e t e r m i n e d as a proportion of total protein l o a d e d using the PDQUEST software package (PDL H u n t ington, NY). Experimental
Time Course
A n i m a l s were anesthetized with halothane mixed with 95% oxygen plus 5% carbon dioxide a n d sacrificed at 12,
Mechanical
and Endothelial
Function
In the control group (n = 6), postischemic recovery (mean percentage of preischemic value) of CO, PAP, a n d CF was 57.0 + 2.8, 63.4 _+ 3.1, a n d 76.6 --_ 3.6, respectively. Postischemic recovery of the coronary r e s p o n s e to 5-HT (mean percentage of preischemic value + s t a n d a r d error of the mean) was 20.3 -+ 3.8. Twenty-four hours after heat shock, postischemic recovery of CO, PAP, CF, a n d coronary flow response to 5-HT (as percentage of preischemic value ± s t a n d a r d error of the mean) was 74.0 ± 2.4, 79.3 ± 5.0, 89.8 ± 1.9, a n d 58.3 ± 7.2, respectively. Similar values (p = not significant) were seen at 26 a n d 30 hours after heat shock. Values o b t a i n e d at 12, 14, 16, 18, a n d 20 hours after heat stress were similar to those in the control a n d s h a m -
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A M R A N I ET A L KINETICS OF INDUCTION OF HEAT-SHOCK PROTEINS
Ann Thorac Surg 1996;61:1407-12
Table 2. Preischemic and Postischemic Mechanical and Endothelial Function at Different Intervals After Heat Stress
Time (h)
CO (mL/min) Pre
PAP (cm H20) Post
12 14 16 18 20 24 26 30 36 48 96
87.5 80.5 74.1 90.3 78.6 77.3 81.3 79.3 69.8 79.3 82.4
± 3.5 + 5.5 ± 5.3 _+ 4.9 _+ 8.2 ± 5.8 ± 7.2 + 9.9 + 5.4 ± 6.6 _+ 8.2
Control Sham
88.0 _+ 5.5 80.6 + 4.7
45.4 46.7 41.4 52.1 45.0 57.2 57.6 62.3 34.2 41.8 46.0
-- 5.4 ± 4.5 ± 7.2 ± 6.3 ± 6.6 ± 4.4 ± 5.1 _+ 5.3 ± 3.8 - 6.3 _+ 6.2
50.2 ÷ 7.0 43.4 ± 6.0
Pre 182.1 184.9 178.2 194.1 174.0 172.3 188.5 196.5 188.8 189.0 173.0
± 8.8 ± 9.1 ± 9.5 + 7.9 ± 6.0 ± 4.9 _+ 6.2 + 9.3 ± 6.5 + 8.9 + 4.9
180.0 ± 8.0 182.0 ± 8.3
CF (mL/min) Post
110.8 114.3 121.0 127.1 104.5 135.8 141.4 158.3 123.6 113.3 117.5
Pre
+ 8.5 ± 6.4 -+ 8.0 + 5.8 ± 8.8 + 5.0 ± 6.2 + 5.9 ± 7.1 _+ 3.9 _+ 5.0
14.7 15.0 13.0 12.7 15.5 13.3 15.4 16.0 12.8 14.7 15.5
117.3 ± 5.0 109.2 ± 7.9
+ 0.9 ± 1.1 _+ 0.5 + 0.7 ± 1.0 + 0.7 _+ 0.9 ± 0.7 _+ 0.9 _+ 0.6 ± 0.5
16.3 + 0.5 15.7 + 0.7
EF (% increase of CF) Post
11.6 10.7 10.7 9.6 12.3 11.0 14.6 14.7 10.6 11.1 12.1
± 0.6 _+ 0.8 -+ 0.6 -+ 0.8 ± 0.3 + 0.4 + 0.7 ± 0.8 ± 0.8 + 0.4 + 0.2
12.5 ± 0.8 12.2 -+ 0.3
Pre 65.5 72.0 57.0 63.5 70.0 59.5 75.0 69.0 74.0 68.8 70.0
± 5.5 _+ 7.0 _+ 5.7 +_ 3.3 ± 9.7 + 5.7 _+ 6.6 ± 8.0 _+ 5.6 _+ 9.3 ± 7.1
74.0 ± 4.7 63.5 ± 7.0
Post 12.2 16.0 11.7 16.5 16.8 34.2 39.0 32.3 18.5 13.5 19.6
± 2.9 ± 5.5 + 4.4 - 5.2 ± 7.0 ± 6.2 + 5.1 _+ 4.9 + 3.9 ± 7.7 _+ 4.1
14.8 ± 7.1 19.8 ± 6.2
a Each v a l u e r e p r e s e n t s t h e m e a n _+ t h e s t a n d a r d e r r o r of t h e m e a n of six hearts. CF - c o r o n a r y flow;
C O = c a r d i a c output;
EF = e n d o t h e l i a l function;
o p e r a t i o n g r o u p s a n d significantly l o w e r t h a n t h o s e obt a i n e d at 24, 26, a n d 30 h o u r s (p < 0.05). Likewise, v a l u e s o b t a i n e d after 30 h o u r s w e r e c o m p a r a b l e to t h o s e in the control a n d s h a m - o p e r a t i o n g r o u p s a n d significantly l o w e r t h a n t h o s e o b t a i n e d at 24, 26, a n d 30 h o u r s (p 0.05). Table 1 a n d F i g u r e 1 s h o w the c o r r e s p o n d i n g p o s t i s c h e m i c r e c o v e r i e s to the t i m e intervals s t u d i e d . Significance is r e p o r t e d in Figure 1. Table 2 s h o w s t h e p r e i s c h e m i c a n d p o s t i s c h e m i c v a l u e s of CO, PAP, CF, a n d c o r o n a r y flow r e s p o n s e to 5-HT.
H S P 70 N o i n d u c t i o n of H S P 70 w a s d e t e c t a b l e in a n i m a l s in the control a n d s h a m - o p e r a t i o n g r o u p s . H e a t - s t r e s s p r o t e i n 70 was d e t e c t a b l e in h e a t - s h o c k e d a n i m a l s f r o m 12 h o u r s after h e a t t r e a t m e n t . P e a k H S P levels w e r e o b s e r v e d at 24 h o u r s a n d d e c l i n e d r a p i d l y after 30 hours. T h e H S P 70 levels, e x p r e s s e d as a p r o p o r t i o n of the total p r o t e i n l o a d e d for e a c h s a m p l e ( m e a s u r e d on a m i d o - s t a i n e d blots), are s h o w n in Table 1. Comment T h e p r e s e n t s t u d y has s h o w n that after p r o l o n g e d cardioplegic arrest, the p o s t i s c h e m i c r e c o v e r y of b o t h cardiac m e c h a n i c a l f u n c t i o n a n d e n d o t h e l i a l f u n c t i o n was significantly i m p r o v e d w h e n t h e i n t e r v a l b e t w e e n h e a t stress a n d i s c h e m i a r a n g e d f r o m 24 to 30 hours. This i n t e r v a l c o r r e l a t e d w i t h the m a x i m a l h e a t - s h o c k res p o n s e as d e t e r m i n e d f r o m levels of the i n d u c i b l e stress p r o t e i n H S P 70. W h e n any cell is s u b j e c t e d to s u b l e t h a l h y p e r t h e r m i a , a v a r i e t y of a d a p t i v e m o d i f i c a t i o n s take place t h a t s e r v e to p r o t e c t the cell f r o m s u b s e q u e n t i n c r e a s e s in t e m p e r ature or o t h e r stresses [1-4]. In a d d i t i o n to h y p e r t h e r m i a , o t h e r stresses i n c l u d i n g i s c h e m i c p r e c o n d i t i o n i n g [13], c h e m i c a l a g e n t s [14], a n d p r e s s u r e o v e r l o a d [15] i n d u c e
PAP
p e a k aortic p r e s s u r e .
H S P synthesis. This s u g g e s t s that t h e r e s p o n s e m a y r e p r e s e n t a g e n e r a l i z e d cellular d e f e n s e m e c h a n i s m a n d h e n c e , the c u r r e n t c o n c e p t of " c r o s s t o l e r a n c e " [6]. Bec a u s e it uses an intrinsic cellular d e f e n s e m e c h a n i s m b e f o r e the v e r y o n s e t of i s c h e m i a , this a p p r o a c h app e a r e d to be a n e l e g a n t t h e r a p e u t i c a l t e r n a t i v e to p r o t e c t against cardiac i s c h e m i a [16]. O v e r the last d e c a d e , the p r o t e c t i v e effect of H S P s against i s c h e m i c d a m a g e has b e e n w i d e l y i n v e s t i g a t e d in v a r i o u s e x p e r i m e n t a l m o d e l s a n d has led to p r o m i s i n g results in m o s t studies. H o w e v e r , s o m e i n v e s t i g a t o r s [6, 13, 17] s t r e s s e d the lack of c o r r e l a t i o n b e t w e e n t h e " p r e s e n c e " of H S P s a n d t h e i r p r o t e c t i v e role. This d i s c r e p a n c y s u g g e s t s that a critical a m o u n t of H S P is n e c e s s a r y to elicit a p r o t e c t i v e effect a n d that this "critical v a l u e " is m o r e i m p o r t a n t to d e f i n e t h a n t h e m e r e p r e s e n c e of HSPs. This is f u r t h e r e m p h a s i z e d by t h e possibility that e a c h p a r t i c u l a r e x p e r i m e n t a l m o d e l (animals, t y p e of stress stimuli, l e n g t h of i s c h e m i a , t y p e of p e r f u s i o n ) m i g h t r e s u l t in different "critical p r o t e c t i v e " c o n c e n t r a t i o n s of HSPs. A t e m p o r a l r e l a t i o n s h i p b e t w e e n the a p p e a r a n c e a n d s u b s e q u e n t d i s a p p e a r a n c e of H S P s w i t h f u n c t i o n a l rec o v e r y has b e e n p r e v i o u s l y r e p o r t e d in n o n q u a n t i t a t i v e H S P studies in t h e i s o l a t e d rat h e a r t s u b j e c t e d to l o w flow i s c h e m i a [10] a n d after r e g i o n a l i s c h e m i a in the rabbit h e a r t [7]. In t h e p r e s e n t study, w e a s s e s s e d the h e a t - s h o c k res p o n s e b y m e a s u r i n g H S P 70 at different i n t e r v a l s r a n g ing f r o m 12 to 96 h o u r s after h e a t stress u s i n g a s e m i q u a n t i t a t i v e m e t h o d . In addition, for e a c h interval, w e e v a l u a t e d the p o s t i s c h e m i c r e c o v e r y of m e c h a n i c a l a n d e n d o t h e l i a l f u n c t i o n after p r o l o n g e d c a r d i o p l e g i c arrest. E n d o t h e l i a l f u n c t i o n was s t u d i e d b e c a u s e in p r e v i o u s work, w e [18] h a v e d e m o n s t r a t e d its i n f l u e n c e on m y o cardial contractility. To date, t h e r e are n o c o m p a r a t i v e d a t a r e g a r d i n g i n d u c t i o n of H S P s in the different cellular c o m p o n e n t s . O u r results s t r o n g l y s u g g e s t that H S P 70 is
Ann Thorac Surg 1996;61:1407-12
i n d u c e d in endothelial cells. This r e m a i n s to be validated in isolated cell preparations. We f o u n d that both maximal levels of HSP 70 as well as optimal recovery of mechanical a n d endothelial function were seen at 24, 26, a n d 30 hours. For all other intervals, the concentration of HSP 70 was significantly lower a n d was associated with a recovery similar to the control group (no heat stress). Thus, it appears that a critical a m o u n t of HSP is necessary to afford protection against ischemia. This study has several limitations, which include the fact that although we have defined the relative a m o u n t of HSP 70 in relation to other proteins in the myocardium, we have not defined the absolute a m o u n t of HSP 70 in the cells concerned. In addition, we have chosen to study one type of HSP (HSP 70), the one that is most c o m m o n l y expressed. Other HSPs could have similar or more important influence on myocardial protection. Finally, the clinical relevance of our results is influenced by the fact that the use of heat stress in the clinical setting is not practical. Other strategies for i n d u c i n g HSPs need to be developed. It is h o p e d that the data presented here will be of value in u n d e r s t a n d i n g a n d possibly applying methods of e n h a n c i n g the intrinsic protective mechanisms of the heart against ischemic damage. We thank Mr Jamal A. Amrani for help with the statistical analysis.
References 1. Hightower LE. Heart shock proteins, chaperones, and proteotoxicity. Cell 1991;66:191-7. 2. Li CG, Werb Z. Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblast. Proc Natl Acad Sci USA 1982;79:3218-22. 3. Sanchez Y, Linquist SL, HSP104 is required for induced thermotolerance. Science 1990;248:1112-5. 4. Marber MS, Latchman DS, Walker JM, Yeilon DM. Cardiac stress protein elevation 24 hours after brief ischemic or heat stress is associated with resistance to myocardial infarction. Circulation 1993;88:1264-72. 5. Hutter MM, Sievers RE, Barbosa V, Wolfe CL. Heat-shock
AMRANIET AL KINETICSOF INDUCTIONOF HEAT-SHOCKPROTEINS
6.
7. 8.
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10. 11. 12.
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15.
16. 17. 18.
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proteins induction in rat hearts. A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection. Circulation 1994;89:355-60. Donnelly TJ, Sievers RE, Vissern FL, Welch WJ, Wolfe CL. Heat-shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion? Circulation 1992;85:769-78. Currie RW, Tanguay RM, Kingma JG. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 1993;87:963-71. Amrani M, Corbett J, Allen NJ, et al. Induction of heat-shock proteins enhances myocardial and endothelial functional recovery after prolonged cardioplegic arrest. Ann Thorac Surg 1994;57:157-60. Amrani M, Allen NJ, O'Shea J, et al. Role of catalase and heat-shock protein on recovery of cardiac endothelial and mechanical function after ischemia. Cardioscience 1993;4: 193-8. Karmazyn M, Mailer K, Currie RW. Acquisition and decay of heat-shock proteins enhanced postischemic ventricular recovery. Am J Physiol 1990;259:H424-31. Neely JR, Leibermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat hearts. Am J Physiol 1967;212:804-14. Amrani M, Ledingham S, Jayakumar J, et al. Detrimental effect on the efficacy of the University of Wisconsin solution when used for cardioplegia at moderate hypothermia. Comparison with the St. Thomas Hospital solution at 4°C and 20°C. Circulation 1992;86(Suppl 2):280-8. Tanaka M, Fujiwara H, Yamasaki K, et al. Ischemic preconditioning elevates cardiac stress protein but does not limit infarct size 24 or 48 hours later in rabbits. Am J Physiol 1994;267:H1476-82. Maulik N, Engelman RM, Wei Z, Lu D, Rousou JA, Das DK. Interleukin-1 alpha preconditioning reduces myocardial ischemia reperfusion injury. Circulation 1993;88(Suppl 2): 387-94. Delcayre C, Samuel JL, Marotte F, Best-Belpomme M, Mercadier JJ, Rappaport L. Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J Clin Invest 1988;82:460-8. Liu X, Engelman RM, Moraru H, et al. Heat shock: a new approach for myocardial preservation in cardiac surgery. Circulation 1992;86(Suppl 2):358-63. Yellon DM, Iliodromitis E, Latchman DS, et al. Whole body heat stress fails to limit infarct size in the reperfused heart. Cardiovasc Res 1992;26:342-6. Amrani M, O'Shea J, Allen N, et al. Role of basal release of nitric oxide on coronary flow and mechanical performance in the isolated rat heart. J Physiol (Lond) 1992;456:681-7.
INVITED COMMENTARY W h e n living cells are subjected to a short elevation in temperature, certain proteins, k n o w n as heat-stress or heat-shock proteins (HSPs), are synthesized. These proteins have b e e n shown to s u b s e q u e n t l y protect the myocyte from not only further heat stress, b u t other metabolic insults, including ischemia/reperfusion injury [1, 2]. The induction of these HSPs has b e e n shown to protect the m y o c a r d i u m from ischemic damage in a n u m b e r of different models a n d species. The effects have i n c l u d e d a reduction in infarct size as d e m o n s t r a t e d by tetrazolium staining, reduced release of creatine kinase, a n d imp r o v e m e n t in contractile functions. Increased levels of HSP have b e e n associated with preservation of tissue adenosine triphosphate after ischemia-reperfusion a n d
decreased production of oxygen-derived free radicals after reperfusion [3]. However, whether one or a combination of these m e c h a n i s m s is mediated through HSP resulting in myocardial protection r e m a i n s u n k n o w n . In the study, A m r a n i a n d associates investigated postischernic recovery of mechanical a n d endothelial cell function in a model of hypothermic cardioplegic arrest a n d correlated this with the synthesis of inducible form of HSP 70. Their results demonstrate that maximal levels of HSP 70 correlated with postischernic recovery observed at 24, 26, a n d 30 hours after hypothermic cardioplegic arrest. For other time intervals, the postischemic functional recovery was comparable with control, although the levels of HSP 70 were still detectable.
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AMRANI ET AL KINETICS OF I N D U C T I O N OF H E A T - S H O C K PROTEINS
This study raises a very important question: what is the critical threshold of HSP 70 to induce protection in the heart? Although the cellular levels of HSP 70 were significantly increased at several time intervals (18, 20, 36, and 48 hours after heat shock), apparently the level of protein was not high enough to protect the myocardium against hypothermic cardioplegic arrest. The results of this study differ somewhat from other studies. For example, Currie and associates [1] reported increased expression of HSP 71 correlated with cardioprotection at 24 hours but not 40 hours after heat shock. A preliminary study by Shipley and colleagues [4] demonstrated rapid induction and accumulation of both HSP 27 and HSP 72 four hours after heat stress. Interestingly, no myocardial protection was afforded until 24 hours after heat stress, and protection was gone by 30 hours after heat stress. The synthesis of HSP after heat stress was rapid, reaching more than 80% of maximum within 4 hours of initial insult. These results suggested that the myocardial protection afforded by heat stress cannot be solely explained on the basis of HSP expression, and may be dependent on posttranslational modification of translocation of HSP, or may be dependent on other as yet unidentified factors. Clearly, there is considerable controversy in this important area, and rigorous studies are required to further elucidate the mechanisms of heat stress induced cardiac protection. Nevertheless, Amrani and colleagues deserve compliments for investigating the role of HSP 70 in this unique model of hypothermic cardioplegic arrest. To borrow a term from our high-tech colleagues, if cardioplegia represented the first generation of cardiac protection, preconditioning represents the second generation. Obviously heat shock, being nonphysiologic, is only a model,
A n n Thorac S u r g 1996;61:1407-12
but the race is on in the laboratory on two fronts: first, the development of clinically applicable preconditioning either metabolically or pharmacologically, and second, the isolation and characterization of these unique HSPs. The prolonged ischemic time associated with cardiac transplantation appears to be a logical initial problem to begin this second generation approach. If a direct cause and effect relationship could be established for expression of HSP 70 versus protection of ischemic myocardium, the protein will open the door to new therapeutic options to reduce damage caused by ischemic heart disease of multiple etiologies.
Rakesh C. Kukreja, PhD Michael L. Hess, MD Eric Lipman Laboratories of Molecular and Cellular Cardiology Division of Cardiology Medical College of Virginia Richmond, VA 23298 References 1. Currie RW, Tanguay RM, Kingma JG Jr. Heat-shock response and limitation of tissue necrosis during occlusion/reperfusion in rabbit hearts. Circulation 1993;87:963--71. 2. Karmazyn M, Mailer K, Currie RW. Acquisition and decay of heat-shock enhanced postischemic ventricular recovery. Am J Physiol 1990;259:H424-31. 3. Mocanu MM, Steare SE, Evans MC, Nugent JH, Yellon DM. Heat stress attenuates free radical release in the isolated perfused rat heart. Free Radic Biol Med 1993;15:459-63. 4. Shipley JB, Qian Y-Z, Levasseur JE, Kukreja RC. Expression of the stress proteins HSP-27 and HSP-72 in rat heart does not correlate with ischemic tolerance after heat shock. Circulation 1995;92(Suppl 1):654.